Development of an Improved Loop-Mediated Isothermal Amplification Assay for On-Site Diagnosis of Fire Blight in Apple and Pear
Fast and accurate diagnosis is needed to eradicate and manage economically important and invasive diseases like fire blight. Loop-mediated isothermal amplification (LAMP) is known as the best on-site diagnostic, because it is fast, highly specific to a target, and less sensitive to inhibitors in sam...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Hanrimwon Publishing Company
2018-06-01
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| Series: | The Plant Pathology Journal |
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| Online Access: | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985645 |
| _version_ | 1819035213563428864 |
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| author | Doo-San Shin Gwang-Il Heo Soo-Hyeong Son Chang-Sik Oh Young-Kee Lee Jae-Soon Cha |
| author_facet | Doo-San Shin Gwang-Il Heo Soo-Hyeong Son Chang-Sik Oh Young-Kee Lee Jae-Soon Cha |
| author_sort | Doo-San Shin |
| collection | DOAJ |
| description | Fast and accurate diagnosis is needed to eradicate and manage economically important and invasive diseases like fire blight. Loop-mediated isothermal amplification (LAMP) is known as the best on-site diagnostic, because it is fast, highly specific to a target, and less sensitive to inhibitors in samples. In this study, LAMP assay that gives more consistent results for on-site diagnosis of fire blight than the previous developed LAMP assays was developed. Primers for new LAMP assay (named as DS-LAMP) were designed from a histidine-tRNA ligase gene (EAMY_RS32025) of E. amylovora CFBP1430 genome. The DS-LAMP amplified DNA (positive detection) only from genomic DNA of E. amylovora strains, not from either E. pyrifoliae (causing black shoot blight) or from Pseudomonas syringae pv. syringae (causing shoot blight on apple trees). The detection limit of DS-LAMP was 10 cells per LAMP reaction, equivalent to 104 cells per ml of the sample extract. DS-LAMP successfully diagnosed the pathogens on four fire-blight infected apple and pear orchards. In addition, it could distinguish black shoot blight from fire blight. The Bühlmann-LAMP, developed previously for on-site diagnosis of fire blight, did not give consistent results for specificity to E. amylovora and on-site diagnosis; it gave positive reactions to three strains of E. pyrifoliae and two strains of P. syringae pv. syringae. It also, gave positive reactions to some healthy sample extracts. DS-LAMP, developed in this study, would give more accurate on-site diagnosis of fire blight, especially in the Republic of Korea, where fire blight and black shoot blight coexist. |
| first_indexed | 2024-12-21T07:46:03Z |
| format | Article |
| id | doaj.art-55fd996d5f6a4653a64c415e5888d761 |
| institution | Directory Open Access Journal |
| issn | 1598-2254 |
| language | English |
| last_indexed | 2024-12-21T07:46:03Z |
| publishDate | 2018-06-01 |
| publisher | Hanrimwon Publishing Company |
| record_format | Article |
| series | The Plant Pathology Journal |
| spelling | doaj.art-55fd996d5f6a4653a64c415e5888d7612022-12-21T19:11:11ZengHanrimwon Publishing CompanyThe Plant Pathology Journal1598-22542018-06-0134319119810.5423/PPJ.FT.03.2018.005510.5423PPJ.FT.03.2018.0055Development of an Improved Loop-Mediated Isothermal Amplification Assay for On-Site Diagnosis of Fire Blight in Apple and PearDoo-San Shin0Gwang-Il Heo1Soo-Hyeong Son2Chang-Sik Oh3Young-Kee Lee4Jae-Soon Cha5Major in Plant Medicine, School of Applied Plant Science & Biotechnology, Chungbuk National University, Cheongju 28644, KoreaMajor in Plant Medicine, School of Applied Plant Science & Biotechnology, Chungbuk National University, Cheongju 28644, KoreaMajor in Plant Medicine, School of Applied Plant Science & Biotechnology, Chungbuk National University, Cheongju 28644, KoreaDepartment of Horticultural Biotechnology, Kyung Hee University, Yongin 17104, KoreaDepartment of Agro-food Safety and Crop Protection, National Institute of Agriculture Sciences, Rural Development Administration, Wanju 55365, KoreaMajor in Plant Medicine, School of Applied Plant Science & Biotechnology, Chungbuk National University, Cheongju 28644, KoreaFast and accurate diagnosis is needed to eradicate and manage economically important and invasive diseases like fire blight. Loop-mediated isothermal amplification (LAMP) is known as the best on-site diagnostic, because it is fast, highly specific to a target, and less sensitive to inhibitors in samples. In this study, LAMP assay that gives more consistent results for on-site diagnosis of fire blight than the previous developed LAMP assays was developed. Primers for new LAMP assay (named as DS-LAMP) were designed from a histidine-tRNA ligase gene (EAMY_RS32025) of E. amylovora CFBP1430 genome. The DS-LAMP amplified DNA (positive detection) only from genomic DNA of E. amylovora strains, not from either E. pyrifoliae (causing black shoot blight) or from Pseudomonas syringae pv. syringae (causing shoot blight on apple trees). The detection limit of DS-LAMP was 10 cells per LAMP reaction, equivalent to 104 cells per ml of the sample extract. DS-LAMP successfully diagnosed the pathogens on four fire-blight infected apple and pear orchards. In addition, it could distinguish black shoot blight from fire blight. The Bühlmann-LAMP, developed previously for on-site diagnosis of fire blight, did not give consistent results for specificity to E. amylovora and on-site diagnosis; it gave positive reactions to three strains of E. pyrifoliae and two strains of P. syringae pv. syringae. It also, gave positive reactions to some healthy sample extracts. DS-LAMP, developed in this study, would give more accurate on-site diagnosis of fire blight, especially in the Republic of Korea, where fire blight and black shoot blight coexist.http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985645applefire blightLAMP assayon-site diagnosispear |
| spellingShingle | Doo-San Shin Gwang-Il Heo Soo-Hyeong Son Chang-Sik Oh Young-Kee Lee Jae-Soon Cha Development of an Improved Loop-Mediated Isothermal Amplification Assay for On-Site Diagnosis of Fire Blight in Apple and Pear The Plant Pathology Journal apple fire blight LAMP assay on-site diagnosis pear |
| title | Development of an Improved Loop-Mediated Isothermal Amplification Assay for On-Site Diagnosis of Fire Blight in Apple and Pear |
| title_full | Development of an Improved Loop-Mediated Isothermal Amplification Assay for On-Site Diagnosis of Fire Blight in Apple and Pear |
| title_fullStr | Development of an Improved Loop-Mediated Isothermal Amplification Assay for On-Site Diagnosis of Fire Blight in Apple and Pear |
| title_full_unstemmed | Development of an Improved Loop-Mediated Isothermal Amplification Assay for On-Site Diagnosis of Fire Blight in Apple and Pear |
| title_short | Development of an Improved Loop-Mediated Isothermal Amplification Assay for On-Site Diagnosis of Fire Blight in Apple and Pear |
| title_sort | development of an improved loop mediated isothermal amplification assay for on site diagnosis of fire blight in apple and pear |
| topic | apple fire blight LAMP assay on-site diagnosis pear |
| url | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985645 |
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